Oxidized cardiolipin and uses to detect cardiolipin antibodies
Inventors
Castro, Arnold R. • Mody, Himanshu Champaklal
Assignees
Arlington Scientific Inc • US Department of Health and Human Services
Publication Number
US-8778619-B2
Publication Date
2014-07-15
Expiration Date
2026-11-17
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Abstract
Compositions, methods and devices for the detection of anti-lipoidal antibodies and the diagnosis of disease, for example, syphilis, are described. In particular, oxidized cardiolipins, which may be conjugated with a variety of attachment molecules, such as BSA, KLH, biotin, synthetic protein MAPS, IgY, streptavidin, or avidin, are described. Such oxidized cardiolipin, alone or complexed with one or more attachment molecules, are useful to detect anti-lipoidal antibodies (such as IgG and IgM antibodies) in subjects, for example, when used in ELISA plates. ELISA plates are described that permit the detection of anti-lipoidal antibodies and that permit the co-detection of nontreponemal and treponemal antibodies in biological samples.
Core Innovation
The invention provides compositions, methods, and devices for detecting anti-lipoidal antibodies and diagnosing diseases such as syphilis. In particular, it focuses on oxidized cardiolipins that can be conjugated with various attachment molecules like BSA, KLH, biotin, synthetic protein MAPS, IgY, streptavidin, or avidin. These oxidized cardiolipins, either alone or complexed with attachment molecules, are useful for detecting anti-lipoidal antibodies, such as IgG and IgM, in subjects, notably when used in ELISA plates. The disclosed ELISA plates enable detection of anti-lipoidal antibodies and facilitate the concurrent detection of non-treponemal and treponemal antibodies in biological samples.
The background problem addressed is the difficulty in detecting anti-lipoidal antibodies, particularly in syphilis diagnosis. Existing non-treponemal tests use naturally occurring cardiolipin but suffer due to cardiolipin's small size and hydrophobic nature, which hinders its attachment to solid supports necessary for immunoassays. Additionally, conjugation of cardiolipin to larger molecules to improve binding has traditionally led to loss of its antigenicity. There is a lack of quantitative immunoassays for non-treponemal IgG and IgM antibodies, and existing rapid tests primarily detect treponemal antibodies. A need exists for a rapid, easy-to-use assay that can concurrently detect both anti-lipoidal and treponemal antibodies to improve syphilis diagnosis and treatment monitoring.
The core innovation enables the attachment of cardiolipin or related lipoidal antigens, modified through oxidation, to solid supports while preserving their antigenicity and specificity for anti-lipoidal antibodies. This is achieved by oxidizing cardiolipin to introduce terminal carboxyl groups on its fatty acid side chains, followed by covalent conjugation to protein carriers or direct attachment to solid supports containing amine groups. The oxidized cardiolipin-protein conjugates can be immobilized on various solid supports including ELISA multi-well plates and lateral flow devices. These supports may also include treponemal antigens to allow simultaneous detection of non-treponemal and treponemal antibodies. The methods permit qualitative and quantitative detection of IgG, IgA, and IgM anti-lipoidal antibodies, offering improved diagnostic accuracy for syphilis, disease staging, and treatment efficacy monitoring.
Claims Coverage
The patent includes multiple independent claims centered on methods of diagnosing syphilis using multi-well plates comprising oxidized cardiolipin. The main inventive features concern the preparation of oxidized cardiolipin, its conjugation and immobilization on solid supports, and the detection of complexes formed with anti-lipoidal antibodies.
Method of diagnosing syphilis using multi-well plates with oxidized cardiolipin
A method comprising contacting a biological sample with a multi-well plate containing oxidized cardiolipin that maintains immunogenicity by oxidation of cardiolipin's fatty acid side chains using periodate and permanganate salts, quenching oxidation with a reducing agent to preserve the β-hydroxyl in the central glycerol, activating carboxyl groups to covalently attach a protein carrier or connect to an amine group on the plate, and detecting complex formation between anti-lipoidal antibodies (IgG, IgA or IgM) and the oxidized cardiolipin to diagnose syphilis.
Enhanced detection via labeled antibodies or cardiolipin
Incorporating a detection step by contacting the solid support with labeled anti-human antibodies (such as labeled goat-anti human IgG or IgM) or labeled cardiolipin to visualize the binding of anti-lipoidal antibodies.
Detection methods utilizing specific labels and antibodies
Specific use of labeled goat-anti human IgG and/or IgM antibodies, alone or in combination, with the label being horseradish peroxidase (HRP) and subsequent use of tetramethyl benzidine (TMB) substrate with spectrophotometric detection at 450 nm.
Multi-well plates incorporating treponemal antigen and oxidized cardiolipin
Methods employing multi-well plates that include both treponemal antigens reacting with T. pallidum antibodies and oxidized cardiolipin immunoreactive with anti-lipoidal antibodies prepared by oxidation and conjugation steps analogous to those above, allowing concurrent detection of both antibody types.
Use of enzyme-linked immunosorbent assay (ELISA) format
The multi-well plate is an ELISA plate used in the diagnostic method for syphilis.
The claims broadly cover methods of preparing oxidized cardiolipin with preserved immunogenicity, conjugating it for solid support attachment, and using such conjugates in multi-well plates, often ELISA plates, to detect anti-lipoidal antibodies for diagnosing syphilis. Additional claims cover detection enhancements using labeled antibodies or cardiolipin and inclusion of treponemal antigens to enable combined antibody detection.
Stated Advantages
Provides an automated, accurate, and quantitative immunoassay for detecting anti-lipoidal antibodies, overcoming difficulties with cardiolipin attachment in prior art.
Enables simultaneous detection of non-treponemal and treponemal antibodies in the same test, facilitating comprehensive syphilis diagnosis.
Allows monitoring of syphilis disease progression and therapy efficacy through quantitative detection of IgG and IgM anti-lipoidal antibodies.
The oxidized cardiolipin conjugates maintain antigenicity while enabling attachment to solid supports, permitting development of rapid, point-of-care immunoassays.
Documented Applications
Detection of anti-lipoidal antibodies (e.g., IgG and IgM) for diagnosis of syphilis.
Monitoring the efficacy of syphilis treatment and disease stage determination.
Use in enzyme-linked immunosorbent assays (ELISA), lateral flow devices, and other immunoassay formats for clinical diagnostics.
Detection of anti-cardiolipin antibodies associated with autoimmune diseases such as lupus.
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